MOLECULAR CHAR OF HOST CELL RECEPTORS FOR TRYPANOSOMA CRUZI

这项提议的长期目标是阐明哺乳动物细胞上作为克氏锥虫受体的分子结构。克氏锥虫是查加斯病的病原体，这种疾病影响着数百万人，导致心脏骤停，并经常伴随死亡。尽管在这一领域取得了重大进展，但对克鲁兹毛滴虫的宿主细胞受体知之甚少。我们发现，纯化的心脏成肌细胞表面74 kDa糖蛋白和该分子的抗体可抑制克氏锥虫结合和内化到心肌成肌细胞。这种糖蛋白与侵袭性的锥虫结合，但不与非侵袭性的表鞭毛虫结合，并且只在可以被锥虫入侵的细胞上表达。克氏锥虫gp83表面转唾液酸酶的重组形式识别这个74 kDa的糖蛋白，它与心脏成肌细胞结合，其kD为1×10-4um，介导锥虫与成肌细胞的结合。识别gp83反式唾液酸酶表位或其重组形式的mAb4A4取消了锥虫与心脏成肌细胞和其他细胞的结合，这是锥虫与宿主细胞结合所必需的。该单抗还取消了4-gp83与74 kDa糖蛋白的结合或可溶性gp83反式唾液酸酶与成肌细胞的结合。鉴于这些发现，我们推测宿主74 kDa糖蛋白可能作为克氏锥虫的受体，介导锥虫结合以促进进入。在这项提议中，我们将检验这一假设，并研究这种受体的分子结构。为此，我们提出了以下的具体目标：a)从心脏细胞的cDNA文库中鉴定表达74 kDa蛋白的克隆，对74 kDa蛋白的全长编码基因进行测序并预测其氨基酸序列，b)表达并纯化重组74 kDa糖蛋白，用于与锥虫的配基结合研究，并评估重组分子及其抗体抑制克氏锥虫感染的能力，c)通过将74 kDa糖蛋白基因导入不被克氏锥虫侵袭的哺乳动物细胞系，测试74 kDa糖蛋白作为克氏锥虫受体的能力。D)为了确定克鲁兹毛滴虫gp83配体上与74 kDa糖蛋白结合的区域，这一提议将产生关于细胞膜蛋白分子结构的新信息和见解，该分子结构可能作为克鲁兹毛滴虫侵袭形式的受体。这些研究将有助于建立宿主细胞识别克氏锥虫以促进锥虫进入的分子基础，这可能对目前仍未治愈的疾病的分子干预具有重要意义。

The long term goal of this proposal is to elucidate the molecular structures on mammalian cells that function as receptors for Trypanosoma cruzi. T. cruzi is the causative agent of Chagas' disease, which affects millions of people causing cardiac arrest, frequently accompanied by death. Despite significant advances in this area very little is known, if any, about host cell receptors for T. cruzi. We have found that the purified surface 74 kDa glycoprotein from heart myoblasts and antibodies to this molecule inhibit T. cruzi trypomastigote binding and internalization into heart myoblasts. This glycoprotein binds to invasive trypomastigote but not to non-invasive epimastigote forms of T. cruzi and is only expressed on cells that can be invaded by trypomastigotes. The recombinant forms of T. cruzi gp83 surface trans-sialidase, which binds to heart myoblasts with a Kd of 1x10-4 muM to mediate trypanosome binding to myoblasts, recognizes this 74 kDa glycoprotein. Binding of trypomastigotes to heart myoblasts and other cells is abolished by mAb 4A4 which recognizes an epitope on the gp83 trans-sialidase or its recombinant form and is required for trypanosome binding to host cells. This monoclonal antibody also abolishes the binding of the 4-gp83 to the 74 kDa glycoprotein or the binding of soluble gp83 trans-sialidase to myoblasts. In view of these findings, we have hypothesized that the host 74 kDa glycoprotein may function as a receptor for T. cruzi to mediate trypanosome binding to facilitate entry. In this proposal we will test this hypothesis and we will investigate the molecular structure of this receptor. To this end, we proposed the following specific aims: a) to identify clones expressing the 74 kDa protein from a cDNA library of heart cells, sequence the full length cDNA encoding the 74 kDa protein and predict its amino acid sequence, b) to express and purify the recombinant 74 kDa glycoprotein for ligand binding studies to trypanosomes, and to assess the ability of the recombinant molecule and its antibodies to inhibit T. cruzi infection, c) to test the ability of the 74 kDa glycoprotein to function as a receptor for T. cruzi by transfecting mammalian cell lines which cannot be invaded by T. cruzi with cDNA coding for the 74 kDa protein, d) to determine the region on the T. cruzi gp83 ligand that binds to the 74 kDa glycoprotein This proposal will generate new information and insights about the molecular structure of a membrane protein of cells that may function as a receptor for invasive forms of T. cruzi. These studies will contributes to establish the molecular basis on T. cruzi recognition by host cells to promote trypanosome entry, which may be important for molecular intervention in yet incurable disease.